New A simplified cell-based assay to identify coronavirus 3CL … · 2020. 8. 29. · 4 60...

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A simplified cell-based assay to identify coronavirus 3CL protease inhibitors 1

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Samuel J. Resnick1,2, Sho Iketani3,4, Seo Jung Hong1, Arie Zask5, Hengrui Liu6, Sungsoo Kim1, 3

Schuyler Melore1, Manoj S. Nair3, Yaoxing Huang3, Nicholas E.S. Tay6, Tomislav Rovis6, Hee Won 4

Yang1, Brent R. Stockwell5,6*, David D. Ho3*, Alejandro Chavez1* 5 6 1 Department of Pathology and Cell Biology, Columbia University Irving Medical Center, New York, 7

NY, 10032, USA 8 2 Medical Scientist Training Program, Columbia University Irving Medical Center, New York, NY, 9

10032, USA 10 3 Aaron Diamond AIDS Research Center, Columbia University Irving Medical Center, New York, NY, 11

10032, USA 12 4 Department of Microbiology and Immunology, Columbia University Irving Medical Center, New 13

York, NY, 10032, USA 14 5 Department of Biological Sciences, Columbia University, New York, NY, 10027, USA 15 6 Department of Chemistry, Columbia University, New York, NY, 10027, USA 16 17

*Correspondence to: [email protected]; [email protected]; 18

[email protected] 19

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.CC-BY-NC-ND 4.0 International licenseavailable under a(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

The copyright holder for this preprintthis version posted August 29, 2020. ; https://doi.org/10.1101/2020.08.29.272864doi: bioRxiv preprint

https://doi.org/10.1101/2020.08.29.272864

http://creativecommons.org/licenses/by-nc-nd/4.0/

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Abstract 21

We describe a mammalian cell-based assay capable of identifying coronavirus 3CL protease 22

(3CLpro) inhibitors without requiring the use of live virus. By enabling the facile testing of compounds 23

across a range of coronavirus 3CLpro enzymes, including the one from SARS-CoV-2, we are able to 24

quickly identify compounds with broad or narrow spectra of activity. We further demonstrate the 25

utility of our approach by performing a curated compound screen along with structure-activity 26

profiling of a series of small molecules to identify compounds with antiviral activity. Throughout these 27

studies, we observed concordance between data emerging from this assay and from live virus 28

assays. By democratizing the testing of 3CL inhibitors to enable screening in the majority of 29

laboratories rather than the few with extensive biosafety infrastructure, we hope to expedite the 30

search for coronavirus 3CL protease inhibitors, to address the current epidemic and future ones that 31

will inevitably arise. 32



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Introduction 33

The outbreak of a novel coronavirus (SARS-CoV-2) infection of the past several months has 34

paralyzed countries around the world1,2. This crisis is further exacerbated by the dearth of approved 35

therapeutics, leaving physicians with few proven treatment options. In the past two decades, the 36

world has already suffered from two other major coronavirus outbreaks, Severe Acute Respiratory 37

Syndrome (SARS) and Middle East Respiratory Syndrome (MERS), suggesting that coronaviruses 38

represent a real and ever-present threat to global health that must be addressed3. Yet, even if 39

therapeutics against the existing epidemic strains are identified, there are several hundred other 40

coronaviruses in active circulation within animal populations, many with the theoretical potential to 41

infect humans. To help identify therapeutics for the current epidemic along with preparing for the 42

next, there is a need for readily deployable small molecule screening assays that enable the 43

identification of therapeutics that are broad-acting across a large collection of coronavirus strains. 44

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During coronavirus infection, the RNA genome is delivered into cells and translated into a pair of 46

polyproteins4. These polyproteins are then processed by a set of virally encoded proteases, of which 47

the three-chymotrypsin-like protease (3CLpro) performs the majority of cleavage events4. As a result 48

of its essential role in viral replication and high degree of conservation across all coronaviruses, 49

3CLpro enzymes represent important targets for therapeutic drug development5,6. Previous work 50

expressing a variety of viral proteases within yeast and mammalian cellular systems have shown 51

that protease expression can lead to profound cellular toxicity, which can be rescued by the addition 52

of protease inhibitors7–12. We hypothesized that if the expression of coronavirus 3CLpro enzymes 53

within mammalian cells leads to a similar toxic phenotype, this could form the basis of an easily 54

implemented mammalian cell-based assay to evaluate protease inhibitors. While multiple assays 55

exist to evaluate protease inhibitors, an assay of the nature we envisioned has clear advantages, as 56

it requires minimal upfront cost or effort, is accessible to many biomedical research labs, does not 57

involve the use of live virus, and requires no specialized reporter to read out protease activity. In 58

contrast, in vitro protease assays using purified protein have formed the backbone of inhibitor 59



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screening, but require upfront efforts to isolate the pure protease and are not conducted under 60

physiologic cellular conditions13,14. In addition, if one desires to identify broad-acting coronavirus 61

inhibitors, one must purify and identify experimental conditions suitable for testing each protease in 62

vitro. An alternative approach for identifying protease inhibitors is the use of live virus which is 63

performed under more biologically relevant conditions, assuming relevant host cell systems can be 64

identified, but requires intensive safety training and specialized biosafety protocols15. For many 65

coronaviruses, no live virus assay exists, limiting the ability to test compounds within mammalian cell 66

systems to a small subset of all coronaviruses16. Furthermore, compounds with activity against live 67

virus may function through a number of mechanisms other than protease inhibition which cannot be 68

readily determined, and may lead to undesired off-target activities which are not realized until much 69

later in the drug development process17,18. 70

71

Here, we report a mammalian cell-based assay to identify coronavirus 3CLpro inhibitors that does 72

not require the use of live virus. We demonstrate the utility of the assay using the SARS-CoV-2 73

3CLpro, with EC50 values obtained from the assay showing good concordance with traditional live 74

virus testing for multiple compounds. We next establish the generality of the approach by testing a 75

diverse set of 3CLpro enzymes. Finally, we perform a small molecule screen, along with structure-76

activity profiling of a set of compounds to find those with enhanced antiviral activity. The presented 77

data support the use of our assay system for the discovery of small molecule 3CLpro inhibitors and 78

the rapid characterization of their activity across multiple coronaviruses to identify those with broad 79

inhibitory activity. 80

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Results 83

Expression of the SARS-CoV-2 3CLpro in HEK293T cells results in cell toxicity and can be 84

used as a readout of drug activity 85



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Prior work has shown that the exogenous expression of viral proteases within cellular systems can 86

result in growth suppression or cell death. Motivated by this work, we sought to determine the effect 87

of expressing the SARS-CoV-2 3CLpro in HEK293T cells. Utilizing a cost-effective crystal-violet-88

based approach to quantify cell abundance, we observed that expression of SARS-CoV-2 3CLpro 89

results in significant growth inhibition as compared to the expression of a control protein, enhanced 90

yellow fluorescent protein (EYFP) (Fig. 1a-b)19. This suppression of growth was dependent upon the 91

catalytic function of the enzyme, as mutating cysteine 145, which is essential for the enzyme’s 92

peptidase activity, abolished the growth defect (Fig. 1a-b). We sought to determine if the observed 93

growth defect could be rescued by incubating cells with GC376, a feline coronavirus inhibitor that 94

was recently reported to have activity against SARS-CoV-2 3CLpro20. In comparison to untreated 95

control cells, the addition of GC376 led to a robust increase in cell growth (Fig. 1c-d). 96

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Compound rescue of transfected 3CLpro cytotoxicity is similar to results obtained with live 98

virus 99

We next tested if this transfection-based assay could be used to determine compound EC50 values 100

and whether the values showed any correlation with those obtained with live virus. After incubating 101

SARS-CoV-2 3CLpro transfected cells with a range of GC376 concentrations, we calculated an EC50 102

of 3.30 µM, which is similar to published values reported using live virus on Vero E6 (EC50 2.2 µM, 103

0.9 µM, 0.18 µM, 4.48 µM) and Vero 76 cells (EC50 3.37 µM) (Fig. 2a and Table 1)20–24. We 104

investigated the assay’s tolerance to deviation by varying the amount of plasmid transfected or the 105

number of cells seeded into wells containing compound (Supplementary Fig. 1). In all cases, the 106

assay was robust to variation, delivering a similar EC50 for GC376 across all conditions. We also 107

tested an orthogonal method of quantifying cell abundance based on fluorescence microscopy and 108

observed agreement with the results obtained with crystal violet staining (Supplementary Fig. 2). 109

This suggests that the assay system is robust to variation and provides consistent results. 110

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We next conducted dose-response profiling for two additional SARS-CoV-2 3CLpro inhibitors, 112

compound 4 and compound 11a, and observed reversal of the toxic effect of the protease in a dose-113

dependent manner (Fig. 2b-c)25,26. In agreement with the results obtained with GC376, the EC50 114

value for compound 4 was comparable to those obtained with live virus, 0.98 µM and 3.023 µM, 115

respectively (Table 1)24. Unexpectedly, we calculated an EC50 of 6.89 µM for 11a, which is 116

approximately 10-fold higher than the literature reported value of 0.53 µM, based on viral plaque 117

assay26. We have noticed that literature reported EC50 values from live virus testing could range over 118

an order of magnitude depending on the exact method employed, as is the case for GC376 (Table 119

1). To resolve this discrepancy between the transfection-based approach and the live virus assay, 120

we conducted live virus testing of 11a using the commonly employed readout of cytopathic effect in 121

Vero E6 cells and observed closer concordance with our transfection-based results (Supplementary 122

Figure 3 and Table 1)20,24,27. To measure the toxicity of each compound, we exposed EYFP-123

transfected cells to each molecule and determined CC50 values (Fig. 2). We also calculated the 124

selectivity index (SI) for each compound tested in this study (Supplementary Table 1). 125

126

We hypothesized that the assay would be able to distinguish between compounds that are only 127

active on the purified SARS-CoV-2 3CLpro and those that are able to inhibit the live virus through 128

protease inhibition. In general, we observe concordance between compounds showing activity within 129

this transfection-based 3CL assay and live virus studies (Supplementary Fig. 4a-e)13. However, 130

within the assay, we did not observe activity for ebselen, a small molecule with demonstrated activity 131

against the SARS-CoV-2 3CLpro in vitro and activity against the SARS-CoV-2 live virus 132

(Supplementary Fig. 4f). We suggest that this may be due to ebselen targeting more than 3CLpro 133

within the live virus assay, which is in line with work showing that ebselen is highly reactive and 134

readily forms selenosulfide bonds with numerous proteins including the SARS-CoV-2 papain-like 135

protease (PLP)18,28,29. 136

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Demonstrating assay compatibility across a range of coronavirus 3CLpro enzymes 138



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We hypothesized that this assay may be used to study other coronavirus 3CLpros to enable users to 139

identify broad-acting inhibitors, as constructs containing other 3CLpro enzymes could be readily 140

synthesized. To test the assay’s generality, we created expression constructs for 3CL proteases 141

from five other coronaviruses (SARS-CoV, MERS-CoV, Bat-CoV-HKU9, HCoV-NL63 and IBV) with 142

variable amino acid identity compared with SARS-CoV-2 3CLpro (Supplementary Fig. 5a). For each 143

of these proteases, we confirmed that expression in mammalian cells resulted in toxicity that is 144

dependent upon the enzyme’s catalytic activity (Supplementary Fig. 5b). Next, we tested GC376, 145

compound 4, and 11a across this panel of proteases. GC376, a drug originally identified for use 146

against the Feline Infectious Peritonitis virus, showed EC50 <10 µM for the most, but not all of 147

proteases tested30. Unexpectedly, compound 4, which was originally designed as a SARS-CoV 148

3CLpro inhibitor showed particular potency against IBV 3CLpro (EC50 = 0.058 µM) along with broad 149

activity (EC50 <10 µM) for all other 3CL proteases tested. In contrast to GC376 and compound 4, 11a 150

had a relatively narrow activity spectrum with EC50 <10 µM against only SARS-CoV and SARS-CoV-151

2 3CLpro enzymes (Fig. 3). Of note, in all cases where previous live virus data was available, the 152

EC50 values obtained from this transfection-based assay were similar (Table 1). 153

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Rapid testing to identify SARS-CoV-2-3CL protease inhibitors 155

Having further determined the assay’s ability to examine the effects of active individual compounds, 156

we sought to determine its suitability for small molecule screening. Before performing the screen, we 157

optimized the testing parameters to ensure suitable performance characteristics (Supplementary Fig. 158

6 and Methods)31. We compiled a collection of 162 diverse protease inhibitors, along with 159

compounds with reported in vitro activity against 3CLpro enzymes or structural similarity to known 160

3CLpro inhibitors (Supplementary Table 2 and Supplementary Table 3). Of the nearly 200 161

compounds tested, two potent hits were identified, GC373 and GRL-0496 (Fig. 4a, Supplementary 162

Table 2)32. 163

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We noted that GC373 is structurally similar to its prodrug GC376, except for the change of the 165

bisulfide salt adduct to an aldehyde warhead22,33. Additional testing of GC373 revealed it to have a 166

similar EC50 as GC376 in both the transfection assay and when tested against live SARS-CoV-2 167

virus, suggesting that the differences in structure has a minimal effect on their potency 168

(Supplementary Fig. 7 and Table 1), although solubility may be affected33. The other hit from the 169

screen, GRL-0496, shares structural similarity to several other compounds within the library, one of 170

which is a previously reported 3CLpro inhibitor (MAC-5576) that failed to show activity against 171

SARS-CoV-2 in a live virus assay24,34. Additional testing of GRL-0496 revealed it to have an EC50 of 172

5.05 µM against SARS-CoV-2 3CLpro within our transfection-based assay (Fig. 4c). To verify GRL-173

0496’s activity, we tested it against live SARS-CoV-2 virus, and confirmed its potency (EC50 = 9.12 174

µM) (Fig. 4d). We next tested GRL-0496 against the full panel of 3CLpro enzymes we had previously 175

examined and observed a narrow range of activity, with EC50 <10 µM only observed against SARS-176

CoV 3CLpro and SARS-CoV-2 3CLpro, in agreement with previous live virus testing (Supplementary 177

Fig. 8 and Table 1)27. We also tested GC373 against the full panel of 3CLpro enzymes and observed 178

concordance with GC376, with SARS-CoV 3CLpro, MERS-CoV 3CL pro, and IBV 3CL pro 179

demonstrating an EC50 <10 µM (Supplementary Fig. 8). 180

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Discussion 182

Given the potential for protease inhibitors in the treatment of viral illnesses, small molecule inhibitors 183

of coronavirus 3CL proteases represent a promising avenue for treating infections caused by this 184

large family of viruses. Here, we present a simplified assay to identify candidate inhibitors under 185

physiologic cellular conditions. This approach presents significant advantages over other methods to 186

detect 3CL protease inhibitory activity with its ease of use and ability to be performed with equipment 187

and reagents commonly available to many biomedical research laboratories. While conventional 188

methods for identifying 3CL protease inhibitors make use of in vitro purified protease, the isolation of 189

sufficiently pure enzyme in its native state can be costly and labor intensive. Furthermore, assays 190

using purified protease fail to consider cell permeability and the influence of the extracellular and 191



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intracellular milieu on compound activity. In comparison to live virus-based assays, the outlined 192

approach does not require extensive biosafety containment. These data also suggest that the 193

approach described here is applicable to a number of coronaviruses for which live virus assays may 194

not be available or would be deemed ethically challenging to be performed even with extensive 195

biosafety infrastructure35,36. Finally, because the phenotype assayed within this approach is driven 196

solely by protease activity, it may enable the distinction between compounds with multiple biological 197

targets and subsequent potential for off-target toxicity from those that function primarily as 3CLpro 198

inhibitors. 199

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A number of surrogate assays that aim to identify molecules with activity against proteins encoded 201

by SARS-CoV-2 have recently been reported37–39. These assays were developed to provide 202

physiologically meaningful molecule testing without needing to use live SARS-CoV-2 cultures in 203

order to allow for rapid testing and widespread adoption to labs without necessary safety 204

infrastructure. The aforementioned assays have focused primarily on identifying neutralizing 205

antibodies targeted at the SARS-CoV-2 spike (S) protein. To our knowledge, a reporter-free 206

surrogate assay to identify coronavirus 3CL protease inhibitors validated with multiple known 207

coronavirus 3CL protease inhibitors has not been reported. 208

209

Within the literature, EC50 values obtained for a 3CLpro inhibitor against live virus can show a broad 210

range of potency, with some compounds demonstrating EC50 values across multiple orders of 211

magnitude (Table 1). These differences appear to be driven by variation in experimental setup such 212

as cell line used, assay readout, incubation period, and initial concentration of virus added. While we 213

have observed agreement between the EC50 values obtained from the described transfection-based 214

method and those reported in the literature, given the differences in EC50 across assays, we suggest 215

caution when comparing results across studies. By developing this transfection-based 3CLpro 216

testing platform, we hope to facilitate the discovery of new coronavirus inhibitors while also 217

facilitating the comparison of existing inhibitors within a single simplified assay system. Furthermore, 218



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we propose that this cellular protease assay system could be industrialized to screen and optimize a 219

large number of compounds to discover potential treatments for future viral pandemics. 220

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Methods 222

Cell Lines and Cell Culture 223

HEK293T and HEK293 cells used in this study were obtained from ATCC. Cells were maintained at 224

37°C in a humidified atmosphere with 5% CO2. HEK293T and HEK293 cells were grown in 225

Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen) which was supplemented with 10% fetal 226

bovine serum (Gibco) and penicillin-streptomycin (Invitrogen). HEK293T and HEK293 cells were 227

confirmed to be free of mycoplasma contamination with the Agilent MycoSensor PCR Assay Kit. 228

To obtain HEK293 cells stably expressing EYFP, cells were co-transfected with EYFP plasmids 229

harboring the piggyBac transposon (pPB bsr2-EYFP) (Yusa et al., 2009) and pCMV-mPBase 230

(mammalian codon-optimized PBase) encoding a piggyBac transposase using Lipofectamine 2000 231

(Invitrogen) according to the manufacturer’s instructions. One day after transfection, the transfected 232

cells were selected with 10 µg/mL of blasticidin (Invitrogen). 233

234

Transfections and Drug Selections 235

24 h prior to transfection, 293T cells were seeded at 65-75% confluency into 24-well plates coated 236

for 30 min with a 1 mg/mL solution of poly-D-lysine (MP Biomedicals Inc.) and washed with PBS 237

(Gibco) once prior to media addition. The next day, 500 ng of 3CLpro expression plasmid, unless 238

otherwise stated, was incubated with Opti-MEM and Lipofectamine 2000 for 30 min at room 239

temperature prior to dribbling on cells, as per manufacturer’s protocol. For plating into drug 240

conditions, 20 h after transfection, cells were washed once with PBS and 200 µL Trypsin-EDTA 241

0.25% (Gibco) was added to cells to release them from the plate. Trypsinized cell slurry was pipetted 242

up and down repeatedly to ensure a single cell suspension. 96-well plates were coated with poly-D-243

lysine, either coated manually with 1 µg/mL poly-D-lysine in PBS solution for 30 min or purchased 244

pre-coated with poly-D-lysine (Corning). Wells were filled with 100 µL of media ± drug and 1 µg/mL 245



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puromycin and were seeded with 9 µL of trypsinized cell slurry. For higher throughput experiments, 246

multiple individually transfected wells of a 24-well plate were combined after trypsinization and prior 247

to seeding in drug. After seeding into wells containing drug and puromycin, cells were incubated for 248

48 h unless otherwise specified. 249

250

Plasmids 251

All vectors used in this study were cloned into the pLEX307 backbone (Addgene #41392) using 252

Gateway LR II Clonase Enzyme mix (Invitrogen). 3CL proteases used in this study were generated 253

using gene fragments ordered from Twist Biosciences. Inactive 3CL proteases were generated by 254

site directed mutagenesis of the essential catalytic cysteine. DNA was transformed into NEB 10-beta 255

high efficiency competent cells. Sanger sequencing to verify proper inserts were done for all 256

plasmids used in this study (Genewiz). 257

258

Plasmid DNA was isolated using standard miniprep buffers (Omega Biotek) and silica membrane 259

columns (Biobasic). To reduce batch-to-batch variability between plasmid DNA isolations and its 260

subsequent impact on transfection efficiency, multiple plasmid DNA extractions were conducted in 261

parallel, diluted to 50 ng/µL and pooled together. 262

263

Crystal Violet Staining and Quantification 264

The crystal violet staining protocol was adapted from Feoktistova et. al.19 Briefly, after compound 265

incubation with 3CLpro expressing cells in 96-well plates, the medium was discarded and cells were 266

washed once with PBS. Cells were incubated with 50 µL of crystal violet staining solution (0.5% 267

crystal violet in 80% water and 20% methanol) and rocked gently for 30 min. The staining solution 268

was removed and cells were washed four times with water using a multichannel pipette. Stained 269

cells were left to dry for ≥4 h on the laboratory bench or within the chemical hood. The crystal violet 270

staining solution was eluted by the addition of 200 µL of methanol over the course of 30 min with 271

gentle rocking. Plates were sealed with parafilm to mitigate methanol evaporation. 100 µL of eluted 272



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stain from each well was transferred to a new 96-well plate for reading in a Tecan Infinite F50 plate 273

reader. Absorbance was measured at 595 nm twice and values were averaged between replicate 274

measurements. Blank wells were included in each batch of experiments, and absorbance values 275

were normalized by background levels of staining from blank wells. 276

277

Statistical Analysis of Dose Response Curves 278

For analysis of crystal violet staining experiments, relative growth was calculated from background 279

normalized absorbance values. Test wells containing drug were divided by average background 280

normalized values from wells where cells were expressing protease and exposed to vehicle, when 281

available. Otherwise, values were normalized by values from protease-expressing cells exposed to 282

the lowest concentration of drug included in the dose-response curve. When there were significant 283

deviations from protease-expressing cells exposed to no drug and protease-expressing cells 284

exposed to lowest concentrations of drug included in the dose-response curve, experiments were 285

repeated with normalization by protease-expressing cells exposed to no drug. CC50 values were 286

calculated in Prism using the nonlinear regression functionality and derived from dose-response 287

curves with EYFP transfected cells. A nonlinear curve fitting function accounting for variable curve 288

slopes was calculated by plotting the normalized response as a function of log(compound). Similarly, 289

EC50 values were calculated in GraphPad Prism also using the nonlinear regression functionality. A 290

nonlinear curve fitting function measuring the stimulatory response of a compound as a function of 291

an unnormalized response was used to calculate the EC50. All reported values were confirmed to not 292

have ambiguous curve fitting. The 95% confidence interval of EC50 calculations was also calculated 293

and included. 294

295

For analysis of live virus experiments, EC50 values were determined by fitting a nonlinear curve to 296

the data with the assumption of a normalized response (GraphPad Prism). Cells were confirmed as 297

mycoplasma negative prior to use. 298

299



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Compound Screening 300

For screening condition optimization, we measured the Z-Factor for replicates of positive controls 301

GC376, tested at 50 µM, and compound 4, tested at 20 µM. Replicate measurements were recorded 302

for DMSO negative controls and positive control compounds after 48, 72, and 96 h of incubation with 303

drug. Background normalized crystal violet absorbance values at each timepoint were collected. 304

305

During the drug screen, within each of the four plates screened, two positive controls wells were 306

included to ensure assay reliability, along with several wells with the negative control 0.1% DMSO 307

condition. All compounds were screened at 10 µM resuspended in DMSO (Fisher Scientific). 308

For hit selection, we employed a robust z-score method. We first normalized data using a robust z-309

score that uses median and median absolute deviation (MAD) instead of mean and standard 310

deviation. We then used a threshold of 3.5 MAD to determine which drugs rescued the cytotoxicity 311

imposed by expression of the viral protease40. 312

313

Live Virus Assay 314

The SARS-CoV-2 strain 2019-nCoV/USA_WA1/2020 was grown and titered in Vero-E6 cells. One 315

day before the experiment, Vero-E6 cells were seeded at 30,000 cells/well in 96 well-plates. Serial 316

dilutions of the test compound were prepared in media (EMEM + 10% FCS + 317

penicillin/streptomycin), pipetted onto cells, and virus was subsequently added to each well at an 318

MOI of 0.2. Cells were incubated at in a humidified environment at 37 °C with 5% CO2 for 72 h after 319

addition of virus. Cytopathic effect was scored by independent, blinded researchers. The reported 320

cytopathic effect value represents the average from two independent reviewers. Percent Inhibition 321

was calculated by comparison to control wells with no inhibitor added. All live virus experiments were 322

conducted in a biosafety level 3 lab. 323

324

Microscopy 325



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Cells plated on a 96-well plate (Greiner Bio-OneTM) and washed once immediately before imaging. 326

EYFP fluorescence imaging was performed using an Axio Observer 7 microscope (Zeiss) equipped 327

with a Plan-Apochromat 10X objective (0.45 N.A.) with 1-by-1 pixel binning. Optical Illumination bias 328

was empirically derived by sampling background areas and subsequently used to flatten images. 329

After a global background subtraction, cell density was calculated based on area of EYFP intensity. 330

331

Compounds and Chemical Synthesis 332

GC376 was purchased from Aobious. Myrecetin, rupintrivir, grazoprevir, saquinavir, fosamprenavir, 333

indinavir, apigenin, quercetin, famotidine, MDL28170, bicailein, betrixaban, and amentoflavone were 334

purchased from Fisher Scientific. Tipranavir was purchased from Cayman Chemical. MAC5576, 335

MAC22272, MAC8120, MAC30731, BTB07404, BTB07408, MWP00332, BTB07417, MWP00508, 336

MWP00333, BTB07407, SPB08384, SPB06613, SPB06636, SPB06591, SPB06593, MWP00709, 337

CC42746, BTB07789, BTB07420, MWP00710, BTB07421, SCR00533, and SEW03089 were 338

purchased from Maybridge. GRL-0496 and GRL-0617 were purchased from Focus Biomolecules. 339

AZVIII-40A (1,2-Benzisothiazol-3(2H)-one) was purchased from Alfa Aesar. Other protease inhibitors 340

in table below were purchased from TargetMol. 341

342

CAS Number Drug Name CAS Number Drug Name 1226781-44-7 Omarigliptin 134381-21-8 Epoxomicin 79183-19-0 Apoptosis Activator 2 110044-82-1 MG101 3731-52-0 Picolamine 125697-93-0 lavendustin C 541-91-3 Muscone 729607-74-3 BMS707035 60-23-1 2-Aminoethanethiol 630420-16-5 Asunaprevir 51146-56-6 Dexibuprofen 76684-89-4 Loxistatin Acid 3416-24-8 Glucosamine 1025015-40-0 GK921 56974-61-9 Gabexate mesylate 292632-98-5 L-685,458 7481-89-2 Zalcitabine 202138-50-9 Tenofovir Disoproxil Fumarate 2016-88-8 Amiloride hydrochloride 937174-76-0 GSK690693 945667-22-1 Saxagliptin hydrate 1256388-51-8 Ledipasvir 668270-12-0 Linagliptin 960374-59-8 ONX0914 486460-32-6 Sitagliptin 1401223-22-0 PI1840 136-77-6 Hexylresorcinol 13552-72-2 (+)-Isocorydine hydrochloride



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497-76-7 Arbutin 697797-51-6 UAMC 00039 dihydrochloride 908-54-3 Diminazene Aceturate 1402727-29-0 PE859

6419-36-9 3-Pyridylacetic acid hydrochloride 847925-91-1 RO4929097

81110-73-8 Racecadotril 254750-02-2 Emricasan 50924-49-7 Mizoribine 161314-82-5 CGS 27023A 7414-83-7 Sodium etidronate 150080-09-4 Talabostat mesylate 103-16-2 Monobenzone 1441674-54-9 Ledipasvir acetone 1180-71-8 Limonin 130370-60-4 Batimastat 472-15-1 Betulinic acid 54857-86-2 TOFA 329-98-6 PMSF 1009734-33-1 HZ1157 42017-89-0 Fenofibric acid 188062-50-2 Abacavir sulfate 196597-26-9 Ramelteon 127373-66-4 Sivelestat 155213-67-5 Ritonavir 1132935-63-7 Dasabuvir 850649-62-6 Alogliptin Benzoate 20575-57-9 Calycosin 179324-69-7 Bortezomib 673-22-3 4-Methoxysalicylaldehyde 546-88-3 Acetohydroxamic acid 111-20-6 Sebacic acid 129618-40-2 Nevirapine 53936-56-4 Deoxyarbutin 192725-17-0 Lopinavir 490-78-8 2-5-dihydroxyacetophenone 39809-25-1 Penciclovir 29700-22-9 Oxyresveratrol 916170-19-9 AOB2796 88321-09-9 Aloxistatin 176161-24-3 Maribavir 864953-29-7 Fostemsavir 1029877-94-8 Trelagliptin succinate 519055-62-0 Tasisulam 1201902-80-8 MLN9708 425386-60-3 Semagacestat 354812-17-2 SC514 35943-35-2 Triciribine 1072833-77-2 Ixazomib 331862-41-0 IMR-1A 871038-72-1 Raltegravir potassium 310456-65-6 IMR1 863329-66-2 PSI6206 210344-98-2 Z-IETD-FMK 82009-34-5 Cilastatin 1624602-30-7 VR23 480-18-2 Taxifolin 161814-49-9 Amprenavir 82956-11-4 Nafamostat mesylate 1312782-34-5 AA26-9 1009119-65-6 Daclatasvir dihydrochloride 1051375-16-6 Dolutegravir 635728-49-3 Darunavir Ethanolate 1026785-59-0 Lomibuvir 142880-36-2 Ilomastat 112246-15-8 Ginsenoside Rh2 697761-98-1 Elvitegravir 256477-09-5 UK371804 1051375-19-9 Dolutegravir sodium 147859-80-1 CA-074 methyl ester 84687-43-4 Astragaloside IV 1404437-62-2 ML281 7770-78-7 Arctigenin 591778-68-6 CP 640186 83-48-7 Stigmasterol 5631-68-5 Hydroumbellic acid 478-01-3 Nobiletin 831-61-8 Ethyl gallate 34157-83-0 Celastrol 2469-34-3 Senegenin 29031-19-4 Glucosamine sulfate 28831-65-4 lithospermic acid



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27409-30-9 Picroside I 209984-56-5 Dibenzazepine 848141-11-7 Alvelestat 209984-57-6 LY411575 128-53-0 N-Ethylmaleimide 1221573-85-8 Paritaprevir 208255-80-5 DAPT 1190307-88-0 Sofosbuvir 865759-25-7 Trelagliptin 1421438-81-4 Crenigacestat 187389-52-2 Z-VAD(OMe)-FMK 1338225-97-0 Doravirine 514-10-3 Abietic Acid 847499-27-8 Delanzomib 229975-97-7 Atazanavir sulfate 25406-64-8 Morroniside

136470-78-5 Abacavir 20633-67-4 Calycosin-7-O-beta-D-glucoside

354813-19-7 Balicatib 59870-68-7 Glabridin 868540-17-4 Carfilzomib 58749-22-7 Licochalcone A 198904-31-3 Atazanavir 1377049-84-7 Velpatasvir 274901-16-5 Vildagliptin 402957-28-2 Telaprevir 244767-67-7 Dapivirine 603139-19-1 Odanacatib 292605-14-2 SB-3CT 206361-99-1 Darunavir 179461-52-0 PD 151746 850876-88-9 Danoprevir 315183-21-2 PAC1 159989-65-8 Nelfinavir Mesylate 59721-29-8 Camostat mesilate 935888-69-0 Oprozomib 154598-52-4 Efavirenz 30827-99-7 AEBSF hydrochloride

1192224-24-0 Des(benzylpyridyl) Atazanavi 273404-37-8 Belnacasan

1194044-20-6 LY2811376 210344-95-9 Z-DEVD-FMK 313967-18-9 FLI06 197855-65-5 Z-FA-FMK 218156-96-8 SRPIN340 149488-17-5 Trovirdine 7497-07-6 NSC 405020 133407-82-6 MG132 103476-89-7 Leupeptin Hemisulfate 1051375-10-0 Cabotegravir 57-11-4 Stearic acid 1146699-66-2 Avagacestat 343

All other compounds used in the study were synthesized and quality checked according to the 344

following protocols. 345

346

347

2-(methylthio)nicotinonitrile (xx-1) The title compound was prepared according to a published 348

procedure; spectral data are in agreement with literature values41. 349

N

CN

SCH3



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1H NMR (500 MHz, CDCl3) δ 8.60 (dd, J = 5.0, 1.8 Hz, 1H), 7.79 (dd, J = 7.7, 1.8 Hz, 1H), 7.07 350

(dd, J = 7.7, 4.9 Hz, 1H), 2.64 (s, 3H). 351

13C NMR (126 MHz, CDCl3) δ 163.7, 152.2, 140.6, 118.4, 115.7, 107.5, 13.4. 352

HRMS High accuracy (ASAP): Calculated for C7H6N2S (M+H)+: 151.0330; found: 151.0327. 353

354

355

2-(methylsulfonyl)nicotinonitrile (xx-2) The title compound was prepared from xx-1 according to 356

a modified procedure from the literature42. 357

358

Pyridine xx-1 (87 mg, 0.58 mmol, 1 equiv) was weighed into 50 mL round bottom flask equipped 359

with a stir bar. The solid was dissolved in 10 mL of anhydrous MeOH, followed by portion-wise 360

(usually 3 portions) additions of mCPBA (500 mg, 2.9 mmol, 5 equiv). Substrate conversion was 361

monitored via TLC analysis (70% EtOAc:Hex to 100% EtOAc), with up to an additional 5 equiv 362

of mCPBA added if necessary. Upon complete conversion of starting material, the reaction was 363

quenched with 20 mL of saturated aqueous NaHCO3, diluted with an additional 20 mL of DCM. 364

The layers were separated and the organic layer was further washed (2x) with 10 mL of of 365

saturated aqueous NaHCO3. The organic layer was then dried in vacuo and purified via silica 366

gel column chromatography (100% EtOAc to 5% MeOH:EtOAc) to yield 51 mg (48% yield) of 367

the desired sulfone as a white solid. 368

1H NMR (400 MHz, CDCl3) δ 8.86 (dd, J = 4.8, 1.6 Hz, 1H), 8.26 (dd, J = 7.9, 1.6 Hz, 1H), 7.70 369

(dd, J = 7.9, 4.8 Hz, 1H), 3.39 (s, 3H). 370

13C NMR (101 MHz, CDCl3) δ 159.8, 151.8, 143.8, 126.8, 113.2, 107.4, 40.1. 371

HRMS High accuracy (ASAP): Calculated for C7H6N2O2S (M+H)+: 183.0228; found: 183.0223. 372

N

CN

S CH3O O



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373

374

2-(methylthio)-6-(trifluoromethyl)nicotinonitrile (xx-3) The title compound was prepared following 375

the published procedure, the product (yellow solid) was carried towards the synthesis of xx-4 376

without further purification41. 377

378

379

2-(methylsulfonyl)-6-(trifluoromethyl)nicotinonitrile (xx-4) The title compound was prepared from 380

xx-3 according to a modified procedure from the literature42. 381

382

Pyridine xx-2 (105 mg, 0.50 mmol, 1 equiv) was weighed into 50 mL round bottom flask 383

equipped with a stir bar. The solid was dissolved in 10 mL of anhydrous MeOH, followed by 384

portion-wise (usually 3 portions) additions of mCPBA (0.86 mg, 5 mmol, 10 equiv). Substrate 385

conversion was monitored via TLC analysis (40% EtOAc:Hex to 60% EtOAc). Upon complete 386

conversion of starting material, the reaction was quenched with 20 mL of saturated aqueous 387

NaHCO3, diluted with an additional 20 mL of DCM. The layers were separated and the organic 388

layer was further washed (2x) with 10 mL of of saturated aqueous NaHCO3. The organic layer 389

was then dried in vacuo and purified via silica gel column chromatography (40% to 80% 390

EtOAc:Hex) to yield 38 mg (30% yield) of the desired sulfone as a white solid. 391

1H NMR (400 MHz, CDCl3) δ 8.49 (dd, J = 8.1, 0.7 Hz, 1H), 8.06 (d, J = 8.1 Hz, 1H). 392

13C NMR (101 MHz, CDCl3) δ 160.4, 149.7 (q, J = 37.6 Hz), 146.0, 123.7 (q, J = 2.3 Hz), 120.0 393

(q, J = 275.5 Hz), 112.1, 109.7, 39.7. 394

N

CN

SCH3CF3

N

CN

S CH3O O

CF3



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19F NMR (376 MHz, CDCl3) δ -68.28. 395

HRMS High accuracy (ASAP): Calculated for C8H5F3N2O2S (M+H)+: 251.0102; found: 251.0104. 396

397

Compound 11a was synthesized according to the specified protocol in Dai. et. al. 202026. Compound 398

11a was confirmed by LCMS with m/z = 453 (M+1) and 451 (M-1). 399

400

Compound 4 was synthesized according to the procedure described in Yang et. al 200625. AZVIII-401

34D was formed as a byproduct (15%) in the synthesis of Compound 4 and was isolated by RP 402

HPLC. 1H NMR (400 MHz, CDCl3) 7.88 (s, 1H), 7.44-7.34 (m, 5H), 6.80 (d, 1H, J = 15.4 Hz), 6.69 (s, 403

1H), 6.16-5.87 (m, 2H), 5.12 (s, 2H), 4.76 (s, 1H), 4.42 (s, 1H), 4.31-3.99 (m, 4H), 3.39 (s, 2H), 2.40-404

1.45 (m, 8H), 1.29–1.20 (m, 12H), 1.13-1.02 (m, 3H), 1.00 – 0.89 (m, 6H). MS M+H = 631. 405

406

AZVIII-38, AZVIII-30, and AZVIII-42 were synthesized according the procedures described in Mou 407

et. al. 200843. AZVIII-37A was synthesized as described in Prior et al. 2013. AZVIII-33B was 408

synthesized according to the method described in Amblard et. al. 201844. 409

410

AZVIII-41A was synthesized according to the procedure described for synthesizing AZVIII-33B in 411

Amblard et. al. 2018 and substituting Z-Leu-OH for BOC-Leu-OH44. 1H NMR (400 MHz, CDCl3) 7.77 412

(s, 1H), 7.41-7.27 (m, 5H), 6.80 (dd, 1H, J = 5.5, 15. 6 Hz), 5.98-5.84 (m, 2H), 5.15-5.03 (m, 2H), 413

4.55 (bd s, 1H), 4.27 (bd s, 1H), 4.23-4.07 (m, 3H), 3.38-3.23 (m, 2H), 2.56–1.40 (m, 8H), 1.33 – 414

1.20 (m, 3H), 0.95 (s, 6H). MS M+H = 474. 415

416

AZVII-43A was synthesized by treatment of a dichloromethane solution of AZVIII-30 and AZVIII-40A 417

with triphenyl phosphine and diethyl azodicarboxylate. 1H NMR (400 MHz, CDCl3) 7.91 (d, 1H, J = 418

8.0), 7.76 (d, 1H, J = 8.0), 7.55-7.34 (m, 2H), 6.16 (bd s, 1H), 5.29 (bd s, 1H), 4.73-4.39 (m, 2H), 419

4.35-3.96 (m, 1H), 3.58-3.16 (m, 2H), 3.79-3.43 (m, 2H), 3.39-3.91 (m, 1H), 1.98-1.77 (m, 1H), 1.73-420

1.57 (m, 1H), 1.42 (s, 9H). MS M+H = 392. 421



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422

423

AZVIII-44B, AZVIII-44D, AZVIII-44E, AZVIII-44H, AZVIII-49C, and AZVIII-49F were synthesized by 424

treatment of the appropriate arylalkylamine or heteroarylalkylamine with the corresponding 425

arylsulfonyl chloride or heteroarylsulfonyl chloride and diisopropylethylamine in dichloromethane. 426

AZVIII-57D was synthesized by treatment of AZVIII-44D with methyl bromoacetate and potassium 427

carbonate in dimethylformamide to give the corresponding ester which was reduced to the 428

corresponding alcohol by treatment with lithium borohydride in tetrahydrofuran. The alcohol was 429

then treated with 1,1,1-tris(acetyloxy)-1,1-dihydro-1,2-benziodoxol-3-(1H)-one and sodium 430

bicarbonate in dichloromethane to give to the corresponding aldehyde AZVIII-57D. AZVIII-57G was 431

synthesized from N-benzyl-4-methoxybenzenesulfonamide according to the procedure used to 432

synthesize AZVIII-57D. 433

434

GC373 was synthesized from GC376 by converting the bisulfite group to an aldehyde group by 435

treatment with aqueous sodium bicarbonate. To a solution of 5.31 mg of GC376 in 200 µL H2O, 2 µL 436

O

O

NH

NHO

NS OO

O

NH

NHO

OH

AZVIII-30

HNS

O+

AZVIII-40A

DEAD

P(Ph)3

AZVIII-43A

CH2Cl2

R NH2 ’R SO2Cl+

R NH

S R’R, R’ = aryl, heteroaryl

N

CH2Cl2 AZVIII-44B AZVIII-44D AZVIII-44E AZVIII-44H AZVIII-49C AZVIII-49F

Br CO2Me

K2CO3

DMF

N S

X

O

O

X = H, OMe

N S

X

OH

LiBH4

THF

N S

X

O

DMP

NaHCO3

CH2Cl2 AZVIII-57D X = HAZVIII-57G X = OMe

O

O

O

O

O

O

O

O



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of saturated NaHCO3 was added. The cloudy mixture was extracted with CH2Cl2, dried over Na2SO4, 437

and concentrated in vacuo to give GC373 as a colorless oil (4.0 mg). 438



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Data availability 439

All reagents generated in this study are without restriction. Plasmids generated in this study will be 440

deposited to Addgene. Source data for all figures are provided with this manuscript online. All 441

statistics were performed using Prism v.8.4.2. 442

443

Acknowledgements 444

Compound 11a was synthesized and generously provided by Shi-Xian Deng, Columbia University. 445

This work was supported by a grant from the Jack Ma Foundation to D.D.H. and A.C. and by grants 446

from Columbia Technology Ventures and the Columbia Translational Therapeutics (TRx) program to 447

B.R.S. Further support for these studies comes from a pilot grant award from the Herbert Irving 448

Comprehensive Cancer Center in partnership with the Irving Institute for Clinical and Translational 449

Research at Columbia University to H.Y. and A.C. A.C. is also supported by a Career Awards for 450

Medical Scientists from the Burroughs Wellcome Fund. S.J.R is supported by NIH grant 451

F31NS111851. S.I. is supported by NIH grant T32AI106711. 452

453

Author contributions 454

S.J.R., S.I, and A.C conceived the project. S.J.R., S.I., B.R.S., D.D.H., and A.C. planned and 455

designed the experiments. S.J.R. performed crystal violet-based assays. S.J.R. and S.K. performed 456

the HEK293-EYFP based assays. S.J.R., S.I., and S.J.H. cloned plasmids. A.Z. and H.L. 457

synthesized compounds and provided compound structure information for synthesized compounds. 458

N.E.S.T. and T.R. synthesized compounds NT-1-21, NT-1-24, and NT-1-32. M.S.N. and Y.H. 459

conducted the live virus assays. S.K. and H.Y. performed imaging and created the HEK293-EYFP 460

cell line. S.J.R. and S.M. conducted data analysis. S.J.R, S.I., S.J.H, and A.C. wrote the manuscript 461

with input from all authors. 462

463

Competing interests 464



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S.I., H.L., A.Z., B.R.S., A.C., and D.D.H. are inventors on a patent application submitted based on 465

some of the molecules described in this work. B.R.S. is an inventor on additional patents and patent 466

applications related to small molecule therapeutics, and co-founded and serves as a consultant to 467

Inzen Therapeutics and Nevrox Limited. 468



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Fig. 1. Expression of SARS-CoV-2 3CLpro in HEK293T cells results in toxicity that can be 572

rescued by GC376. a. SARS-CoV-2 3CL toxicity is dependent on protease activity and can be 573

visualized with crystal violet staining. b. Quantification of crystal violet staining in a. c. Treatment of 574

SARS-CoV-2 3CLpro expressing cells with protease inhibitor GC376 results in rescue of cytotoxicity. 575

d. Quantification of c. Data are shown as mean ± s.d. for four technical replicates. 576

577

a b

c d

EYFP

SARS-CoV-2 3CLpro

SARS-CoV-2 3CLpro C145A

SARS-CoV-2 3CLpro + Vehicle

SARS-CoV-2 3CLpro + 50 µM GC376

EYFP

SARS-CoV-2

3CLpro

SARS-CoV-2

3CLpro

C14

5A0.0

0.2

0.4

0.6

Abs

orba

nce

Vehicle 50µM GC3760.0

0.2

0.4

0.6

Abs

orba

nce



https://doi.org/10.1101/2020.08.29.272864


29

Fig. 2. Dose response curves for SARS-CoV-2 3CLpro can be conducted with transfection-578

based assays. a-c. SARS-CoV-2 3CLpro can be inhibited by known 3CLpro inhibitors GC376, 579

compound 4, and 11a. The toxicity of each compound was determined by treating EYFP-transfected 580

cells with indicated concentrations of drug and is reported as Cell Viability. d. Chemical structures for 581

each of the compounds tested. EC50 values are displayed as best-fit value alongside 95% 582

confidence interval. CC50 values are displayed as best-fit value. Data are shown as mean ± s.d. for 583

four technical replicates. 584

585

-6 -4 -2 0 2 40

1

2

3

4

0

50

100

150

log[GC376], µM

Rel

ativ

e G

row

th

EC50 = 3.30 µM95% CI: [1.81 - 6.04]

CC50 = >100 µM

Cell V

iability (%)

a b

c

-6 -4 -2 0 2 40

1

2

3

4

0

50

100

150

log[Compound 4], µM

Rel

ativ

e G

row

th

EC50 = 0.98 µM95% CI: [0.48 - 2.01]

CC50 = 81 µMRelative Growth

Cell Viability

Cell V

iability (%)

GC376 Compound 4

11a

d

-6 -4 -2 0 2 40

1

2

3

4

0

50

100

150

log[11a], µM

Rel

ativ

e G

row

th

EC50 = 6.89 µM95% CI: [3.80 - 12.59]

CC50 = 48 µM

Cell V

iability (%)



https://doi.org/10.1101/2020.08.29.272864


30

Fig. 3. The activity of GC376, compound 4, and 11a show variable effectiveness and potency 586

against the coronavirus 3CL proteases from SARS-CoV, MERS-CoV, Bat-CoV-HKU9, HCoV-587

NL63, and IBV. EC50 values are displayed as best-fit value alongside 95% confidence interval. CC50 588

values are displayed as best-fit value. Data are shown as mean ± s.d. for three or four technical 589

replicates. 590



https://doi.org/10.1101/2020.08.29.272864


31

591

GC376 Compound 4 11aSA

RS-

CoV

3CLp

roM

ERS-

CoV

3CLp

roB

at-C

oV-H

KU

9 3C

Lpro

HC

oV-N

L63

3CLp

roIB

V 3C

Lpro

-6 -4 -2 0 2 40

1

2

3

0

50

100

150

log[11a], µM

Rel

ativ

e G

row

th

Cell V

iability (%)

-6 -4 -2 0 2 40

1

2

3

0

50

100

150

log[11a], µM

Rel

ativ

e G

row

th

Cell V

iability (%)

-4 -2 0 2 40

1

2

3

4

0

50

100

150

log[GC376], µM

Rel

ativ

e G

row

thEC50 = 5.83 µM

95% CI: [3.58 - 9.38] Cell V

iability (%)

-6 -4 -2 0 2 40

1

2

3

4

5

0

50

100

150


Rel

ativ

e G

row

th

EC50 = 3.17 µM95% CI: [2.03 - 4.99] C

ell Viability (%

)

-6 -4 -2 0 2 40

1

2

3

0

50

100

150

log[11a], µM

Rel

ativ

e G

row

th

EC50 = 5.38 µM95% CI: [1.63 - 32.5]

Cell V

iability (%)

-4 -2 0 2 40

1

2

3

0

50

100

150

log[GC376], µM

Rel

ativ

e G

row

th

EC50 = 7.44 µM95% CI: [4.01 - 14.04] C

ell Viability (%

)

-6 -4 -2 0 2 40

1

2

3

4

0

50

100

150


Rel

ativ

e G

row

th

EC50 = 1.40 µM95% CI: [0.78 - 2.48]

Cell V

iability (%)

-4 -2 0 2 40

1

2

3

0

50

100

150

log[GC376], µM

Rel

ativ

e G

row

th

EC50 = 11.07 µM95% CI: [6.07 - 20.9] C

ell Viability (%

)

-6 -4 -2 0 2 40

1

2

3

0

50

100

150


Rel

ativ

e G

row

th

EC50 = 4.11 µM95% CI: [2.18 - 7.86]

Cell V

iability (%)

-4 -2 0 2 40

1

2

3

4

0

50

100

150

log[GC376], µM

Rel

ativ

e G

row

th

EC50 = >50 µM

Cell V

iability (%)

-6 -4 -2 0 2 40

1

2

3

4

0

50

100

150


Rel

ativ

e G

row

th

EC50 = 4.92 µM95% CI: [3.53 - 6.85]

Cell V

iability (%)

-6 -4 -2 0 2 40

1

2

3

0

50

100

150

log[11a], µM

Rel

ativ

e G

row

th

EC50 = >20 µM

Cell V

iability (%)

-4 -2 0 2 40

1

2

3

0

50

100

150

log[GC376], µM

Rel

ativ

e G

row

th

EC50 = 0.58 µM95% CI: [0.27 - 1.21]

Cell V

iability (%)

-6 -4 -2 0 2 40

1

2

3

4

0

50

100

150


Rel

ativ

e G

row

th

EC50 = 0.058 µM95% CI: [0.028 - 0.1]

Cell V

iability (%)

-6 -4 -2 0 2 40

1

2

3

0

50

100

150

log[11a], µM

Rel

ativ

e G

row

th

EC50 = >20 µM

Cell V

iability (%)



https://doi.org/10.1101/2020.08.29.272864


32

Fig. 4. Small-scale drug screen and structure-activity profiling at 10 µM identify two 592

compounds, GC373 and GRL-0496, with activity against the SARS-CoV-2 3CLpro. a. 593

Identification of hits from the drug screen and structure-activity profiling. Positive control compounds 594

were included in each plate and are highlighted. b. Compounds with structural similarity to known 595

inhibitors. Compounds in bold are molecules that show activity against the SARS-CoV-2 3CLpro at 596

10 µM. c. Dose-response profiling and cytotoxicity determination of GRL-0496 against the SARS-597

CoV-2 3CLpro. d. Live virus testing of GRL-0496 against SARS-CoV-2. 598

EC50 values are displayed as best-fit value alongside 95% confidence interval. The live virus assay 599

was conducted with two biological replicates, each with three technical replicates and the EC50 value 600

was derived from all replicates. CC50 values are displayed as best-fit value. Data are shown as mean 601

± s.d. for three or four technical replicates. 602



https://doi.org/10.1101/2020.08.29.272864


33

603

a

MAC-5576 BTB07408 BTB07417GRL-0496

Compound 4 AZVIII-34D

GC376 AZVIII-41A GC373

b

c

-3 -2 -1 0 1 2

0

50

100

log[GRL-0496], µM

% S

AR

S-C

oV-2

Inhi

bitio

n

EC50 = 9.12 µM95% CI: [7.00 - 11.88]

d

-6 -4 -2 0 2 4

1

2

0

50

100

150

log[GRL-0496], µM

Rel

ativ

e G

row

th

EC50 = 5.05 µM95% CI: [2.99 - 8.56]

CC50 = 81 uM Cell V

iability (%)

-20

0

20

40

60

Rob

ust Z

Sco

re

Plate 1 Plate 2 Plate 3 Plate 4

GRL-0496

GC376 - 50 µMCompound 4 - 20 µM

GC373 Controls

Compound 4 - 10 µM



https://doi.org/10.1101/2020.08.29.272864


34

Table 1. Comparison of literature reported live virus based EC50 values compared to values 604

generated during this study. CPE = Cytopathic effect. 605

Protease Drug Calculated

EC50 (µM)

Literature

Reported

Value (µM)

Method Cell

Line

Citation

SARS-CoV-2

3CLpro

GC376 3.3 3.37 CPE Vero 76 Ma et. al.

0.9 Plaque

Assay

Vero E6 Vuong et. al.

0.18 qPCR Vero E6 Luan et. al.

2.2 qPCR Vero E6 Froggatt et. al.

4.48 CPE Vero E6 Iketani et. al.

SARS-CoV-2

3CLpro

11a 6.89 2.06 CPE Vero E6 This study

0.53 Plaque

Assay

Vero E6 Dai et. al.

SARS-CoV-2

3CLpro

Compound

4

0.98 3.023 CPE Vero E6 Iketani et. al.

SARS-CoV-2

3CLpro

GRL-0496 5.05 9.12 CPE Vero E6 This study

SARS-CoV

3CLpro

GRL-0496 7.84 6.9 CPE Vero E6 Ghosh et. al.

SARS-CoV-2

3CLpro

GC373 2.8 1.5 Plaque

Assay

Vero E6 Vuong et. al.

4.83 CPE Vero E6 This Study

606



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